Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-11-05
2004-01-27
Martinell, James (Department: 1633)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S254200, C435S325000, C435S352000, C435S320100, C435S069400, C536S023500, C536S023510, C536S023100
Reexamination Certificate
active
06682910
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the field of genetic engineering, particularly to the expression of glycoprotein products of recombinant genes, and more particularly to the expression of high levels of biologically active human erythropoietin from stably transfected cells.
BACKGROUND OF THE INVENTION
The hormone erythropoietin plays a major role in regulating erythropoiesis, the formation of red blood cells, and deficiencies of erythropoietin result in anemia. Detailed studies of the hormone and attempts at replacement therapy have been difficult due to the scarcity of purified material.
Normal production of human red blood cells requires the secretion of erythropoietin by the kidney, apparently as the mature glycoprotein. In the steady state this hormone circulates in the blood at a concentration of 10 to 18 milliunits (128-230 picograms) per milliliter, and with the stimulus of severe tissue hypoxia (oxygen deficiency) the levels may increase as much as 1000-fold. The elevated hormone level triggers proliferation and differentiation of a population of receptive stem cells in the bone marrow, stimulates hemoglobin synthesis in maturing erythroid cells, and accelerates release of red cells from the marrow into circulation, thereby increasing the red cell mass and ameliorating the hypoxic conditions. Patients with deficiencies of erythropoietin, such as those with chronic renal failure, often suffer severe anemia.
Erythropoietin is a glycoprotein of 34-38 kd with approximately 40% of its molecular weight provided by carbohydrate. At least one disulfide bridge is required for activity. Little is known about the structure of this hormone, and the details of its synthesis are not well understood. Recent isolations of cDNA and genomic clones provide opportunities to analyze control of erythropoietin production, but the expression of biologically active human erythropoietin in sufficient quantities for replacement therapy has not been achieved.
SUMMARY OF THE INVENTION
Pursuant to this disclosure, biologically active human erythropoietin can be expressed at high levels (at nominal titers exceeding two million Units per liter of supernatant) from stably transfected mammalian cell lines. Thus, an abundant source of purified human erythropoietin for clinical applications is provided. Surprisingly high expression of erythropoietin is achieved by transfecting host cell lines with the Apa I restriction fragment of the human erythropoietin gene. The sense strand of the Apa I restriction fragment has a nucleotide sequence corresponding to that shown in FIG.
1
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REFERENCES:
patent: 4377513 (1983-03-01), Sugimoto et al.
patent: 4703008 (1987-10-01), Lin
patent: 4751084 (1988-06-01), Feder et al.
patent: 4835260 (1989-05-01), Shoemaker
patent: 5441868 (1995-08-01), Lin
patent: 5688679 (1997-11-01), Powell
patent: 0 255 231 (1988-02-01), None
patent: 85/02610 (1985-06-01), None
Powell et al, Proc. Natl. Acad. Sci. USA 83: 6465 (1986).*
Tsuda et al, Eur. J. Biochem. 188: 405 (1990).*
Lin et al., “Cloning and Expression of the Human Erythropoietin Gene”, Proc. Nat'l. Acad. Sci., vol. 82, No. 22, 7580-7584 (1985).
Jelkmann, “Renal Erythropoietin: Properties and Production”, Rev. Physiol. Biochem. Pharmacal., vol. 104, 140-149 (1986).
Werner et al., “Purification of Proteins Produced by Biotechnological Process”, Arzneim./Forsch./Drug Res., vol. 38, No. 3, 422-428 (1988).
Congote, “Effects pf Erythropoietin on Uridine Metabolism in Cell Cultures of Fetal Calf Liver”, Can. J. Biochem. Cell Biol. vol. 62, Nos. 2 and 3, 143-149 (1984).
Krystal et al., “GM Affi-Gen Blue Chromatography of Human Uridine: A Simple One-Step Procedure for Obtaining Erythropoietin Suitable for in Vitro Erythropoietin Progenitor Assays”, British Journal of Hematology, vol. 58, No. 3, 533-546 (1984).
Broudy et al., “Recombinant Human Erythropoietin: Purification and Analysis of Carbohydrate Linkage”, Archives of Biochemistry and Biophysics, vol. 265, No. 2, 329-336 (1988).
Quelle et al., “High-Level Expression and Purification of a Recombinant Human Erythropoietin Produced Using a Baculovirus Vector,” Blood, vol. 74, No. 2, 652-657 (1989).
Yanagi et al., “Recombinant Human Erythropoietin Produced by Namalwa Cells”, DNA, vol. 8, No. 6, 419-427 (1989).
Lee-Huang et al. “The Human Erythropoietin-Encoding Gene Contains a CAAT box, TATA boxes and Other Transcriptional Regulatory Elements in its 5′ Flanking Region”, Gene, vol. 128, No. 2, 227-236 (1993).
Thompson et al., Induction of Tyrosine &agr;-Ketoglutarate Transminase by Steroid Hormones in a Newly Established Tissue Culture Cell Line, Proc. Nat'l. Acad. Sci., vol. 56, No. 1, 296-303 (1966).
Busby et al., “Expression of Active Human Factor IX in Transfected Cells”, Nature, vol. 316, No. 6025, 271-273 (1985).
Nimtz et al., Structures of Sialylated Oligosaccharide of Human Erythropoietin Expressed in Recombinant BH-21 Cells:, Eur. J. Biochem. vol. 213, No. 1, 39-56 (1993).
Interference No. 102, 334, Fritsch V. Lin, Board of Patent Appeals and Interferences, 1739-1742 (1992).
McDonald et al., “Recovery of Thrombopoietin During Purification”, Biochemical Medicine and Metabolic Biology, vol. 37, 335-343 (1987).
Baig, “Purification of Human &agr;-Fetoprotein”, Analytical Biochemistry, vol. 101, 200-203 (1980).
Hearn, “General Strategies in the Separation of Proteins by High Performance Liquid Chromatographic Methods”, Journal of Chromatography, vol. 418, 3-26 (1987).
Sytkowski et al., Biological Activity and Structural Stability of N-Deglycosylated Recombinant Human Erythropoietin, Biochemical/and Biophysical Research Communications, vol. 176, No. 2, 698-703 (1991).
Miyake et al., “Purification of Human Erythropoietin”, Journal of Biological Chemistry, vol. 252, No. 15, 5558-5564 (1977).
Nimtz et al., “Identification and Structural Characterization of a Mannose-6-Phosphate Containing Oligomannosidic N-Glycan from Human Erythropoietin Secreted by Recombinant BHK-21 Cells”, FEBS Letters, vol. 365, 203-208 (1995).
Maniatis et al., “Molecular Cloning: A laboratory Manual”, 1982.
Berger “Methods in Enzymology”, Guide to Molecular Cloning Techniques, vol. 152, 696-697, 1987.
New England Biolabs Catalog, 1995 p. 14.
Martinell James
Senniger Powers Leavitt & Roedel
University of Washington
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